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Metagenomic Analysis of Biofilms in Drinking Water by Gaelin Kopec-Belliveau
Overview of the Study The majority of naturally occurring biofilms contain many microorganisms that have not yet been cultured, due to this there is very little known about their genetic information. This prompted a study to investigate the metagenome of biofilms growing on rubber-coated valves in common drinking water systems. Three different methods were employed in this study. The first was a sequence analysis done on 650 16S rRNA clones found in the biofilms. This indicated an extremely high diversity in the biofilms. The second method used involved snapshot gene sequencing. This meant comparative searching for roughly 5,000 random sequence clones taken from the biofilms in public databases. This resulted in the identification of 1,026 functional groups that were run through a similarity analysis. The final strategy used was the combination of the DNA sequence information found to create definitive protein sequences from whole-cosmid sequencing. The sequence information generated was then used to make a database with all the phylogenetic and genomic information on microbial biofilm communities in common drinking water systems. Thankfully, none of the DNA or protein sequences identifies could be directly liked to any pathogenetic traits.1 Materials and Methods The biofilms sampled for this study were located on rubber-coated valves in drinking water networks from a town in northwestern Germany. Before the microbes were removed from the valves they were submerged in non chlorinated drinking water for 4 to 7 months. Samples were collected directly from the surface of the valves and nucleic acids were extracted directly from the biofilms. From these biospecimens, cosmic and small insert libraries were constructed. In order to construct cloned 16S rRNA sequences, ribosomal DNAs from the biofilms were amplified by PCR from DNA and run through a series of methods to produce plasmid DNAs containing rRNA inserts that could then be sequenced. To make sense of this rRNA, the sequences were compared to reference sequences contained in the NCBI nucleotide sequence database. The sequences were also screened for chimeres using the Check_Chimera program of the Ribosome Database Project. Additionally, the sequences were split into 5' and 3' fragments and each was analyzed by BLAST searching of GenBank. Chimeres found during this screening were removed from the data set. This allowed the sequences to be grouped into phylogenetic divisions. A dendrogram of the sequences was constructed to illustrate similar clusters. To do this, 16S rRNA gene sequences of the main bacterial divisions was aligned with the cloned sequences from the biofilm samples.1 Results and Conclusions Based on the data collected from the three biofilms on the rubber-coated valves, the phylogenetic diversity of the bacterium biofilm community was assessed. The most common bacterial phylotypes found in the biofilms were alpha-, delta-, and gamma-''Proteobateria, Actinobacteria, and the ''Cytophaga-Flavobacterium-Bacteroids group. Roughly 86% of the bacteria found were Proteobacteria, making this the largest represented group of microbes in the biofilm community. The other two groups constituted a small percentage of the remaining bacteria. Additionally, the final percentage of the bacteria in the community were found to be highly similar to unclassified bacteria. A very small number of the sequences identified were closely related to microbes in the genera Rhizobium and Bradyhizobium. Based on the results it was found that extremely limited pathogenetic potential was present in these biofilms, indicating that the resident microorganisms found in public drinking water systems are not a threat. The data collected was also used to set up a publicly accessible database to enable further investigation into the complex diversity of biofilms in drinking water.1 References 1 C. Schmeisser, C. S., C. Raasch, J. Wingender, K.N. Timmis, D.F. Wenderoth, H.-C. Flemming, H. Liesegang, R.A. Schmitz, K.-E. Jaeger, W.R. Streit. (2003). Metagenome Survey of Biofilms in Drinking-Water Networks. Applied Environmental Microbiology 69(12), 7298-7309.